Journal: iScience

Article Title: A calcium-sensing receptor dileucine motif directs internalization to spatially distinct endosomal signaling pathways

doi: 10.1016/j.isci.2025.112651

Figure Lengend Snippet: CaSR traffics to Rab4 pathways and recycles by Rab4 and Rab11 (A and B) BRET between Venus-Rab4 and CaSR-Nluc-WT or CaSR-Nluc-AA ( N = 7), or CaSR-Nluc-WT in HEK-AP2σ-WT or HEK-AP2σ-R15H cells ( N = 9), with (B) AUC. (C) BRET between Rab4-Venus and CaSR-Nluc-WT, with vehicle (DMSO) or Dyngo-4a. N = 5. (D) SIM of SNAP-CaSR-647 and Venus-Rab4. Scale, 5 μm. Arrows show colocalization. N = 23 (0 mM), N = 22 (3 mM) from N = 4 biological replicates. (E) SIM images of cells exposed to FLAG antibody and either non-permeabilized (total) or exposed to 0 or 3 mM Ca 2+ e for 30 min or 30 min with 3-h recovery, before acid strip to remove cell surface FLAG. Internalization occurs in cells stimulated with 3 mM Ca 2+ e conditions other than plates incubated at 4°C to block endocytosis. Cells transfected with Rab4 siRNA and Rab11-DN have reduced receptor recycling (seen as FLAG expression at cell surfaces) than cells transfected with scrambled siRNA or Rab11-WT. Scale, 5 μm. (F) Non-permeabilized cells incubated with FLAG antibody and exposed to 0 or 3 mM Ca 2+ e for 30 min or 30 min with 3-h recovery to monitor recycling. Comparisons to 3 mM (30 min) in black, to 3 mM (30 min with recovery) in red, and to 0 mM (30 min with recovery) in blue. N = 5. (G) Antibody feeding in cells transfected with scrambled siRNA, Rab4 siRNA, or Rab11a-dominant negative (DN). Comparisons to 0 min (blue). N = 5. (H) Antibody feeding assays in which permeabilized cells were incubated with FLAG antibody and exposed to 0 or 3 mM Ca 2+ e for 30 min or 30 min with 3-h recovery. Statistics show comparisons to 3 mM (30 min) in black, to 3 mM (30 min with recovery) in red, and to 0 mM (30 min with recovery) in blue. N = 5. (I) Antibody feeding assays in cells transfected with scrambled siRNA, Rab4 siRNA, or Rab11a-DN and permeabilized to monitor total CaSR. Statistical analyses show comparisons to 0 min in blue and between 0 and 3 mM in each condition in black. N = 4. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple-comparisons test for (B), unpaired t test in (C), and one-way ANOVA with Sidak’s multiple-comparisons test in (F)–(I). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. Data shows mean + SEM in A–C,and F–I.

Article Snippet: Rab4 (RAB4A) Human siRNA Oligo Duplex (Locus ID 5867) , Origene , Catalog. no. SR303946.

Techniques: Stripping Membranes, Incubation, Blocking Assay, Transfection, Expressing, Dominant Negative Mutation

Journal: iScience

Article Title: A calcium-sensing receptor dileucine motif directs internalization to spatially distinct endosomal signaling pathways

doi: 10.1016/j.isci.2025.112651

Figure Lengend Snippet: CaSR signaling from Rab4- and Rab9-positive endosomes contributes to sustained signaling (A) BRET between Venus-Rab4 and Nluc-mGq in HEK-CaSR. Calcium ( N = 9), spermine ( N = 6). (B) Ca 2+ -induced BRET with vehicle (DMSO) or Dyngo-4a. N = 8. (C and D) BRET between Venus-Rab4 and CaSR-Nluc-WT or CaSR-Nluc-AA or (D) CaSR-Nluc-WT in HEK-AP2σ-WT or HEK-AP2σ-R15H cells. N = 7. (E) AUC of ligand-induced BRET between Nluc-GRK3 and Venus-Gβγ in HEK-CaSR overexpressing Gα11, with scrambled or Rab4 siRNA. N = 8. (F) AUC of NanoBiT IP 3 responses with scrambled or Rab4 siRNA. N = 6. (G) Western blot of p -ERK1/2 in HEK-CaSR with scrambled or Rab4 siRNA following a 5-min pulse of 3 mM Ca 2+ then 0.1 mM Ca 2+ . Quantification of densitometry from five western blots is shown below. (H) BRET between Venus-Rab9 and Nluc-mGq in HEK-CaSR. Calcium ( N = 9), spermine ( N = 6). (I) BRET with vehicle (DMSO) or Dyngo-4a. N = 8. (J and K) Ca 2+ -induced BRET in (J) cells transfected with CaSR-Nluc-WT or CaSR-Nluc-AA ( N = 5) or in (K) HEK-AP2σ-WT or HEK-AP2σ-R15H cells ( N = 10). (L) BRET between Nluc-GRK3 and Venus-Gβγ in HEK-CaSR overexpressing Gα11, with scrambled or Rab9 siRNA. N = 8. (M) NanoBiT IP 3 responses with scrambled or Rab9 siRNA. N = 6. (N) p -ERK1/2 responses with scrambled or Rab4 siRNA and exposed to a 5-min pulse of 3 mM Ca 2+ e . Quantification of densitometry from four western blots is shown below. Data shows mean ± SEM. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. Black asterisks show vehicle vs. agonist. Statistical analyses were performed using one-way ANOVA with Sidak’s multiple comparisons test for (A), (B), (C), (D), (G), (H), (I), (J), (K), and (N), and two-way ANOVA with Sidak’s test for (E), (F), (L), and (M).

Article Snippet: Rab4 (RAB4A) Human siRNA Oligo Duplex (Locus ID 5867) , Origene , Catalog. no. SR303946.

Techniques: Western Blot, Transfection

Journal: iScience

Article Title: A calcium-sensing receptor dileucine motif directs internalization to spatially distinct endosomal signaling pathways

doi: 10.1016/j.isci.2025.112651

Figure Lengend Snippet: Cinacalcet increases CaSR signaling in FHH3-associated AP2σ-R15H mutant cells by enhancing sustained signaling (A) BRET between Venus-Rab4 and CaSR-Nluc with vehicle or cinacalcet. (B and C) Dose response of BRET responses with increasing cinacalcet concentrations with (C) pEC50 values. N = 4. (D and E) BRET between Venus-Rab4 and CaSR-Nluc with vehicle or NPS-2143, with (E) dose response. N = 6. (F and G) BRET between Venus-Rab4 and Nluc-mGq with increasing cinacalcet concentrations, with (G) pEC50. N = 4. (H) BRET between Nluc-mGq and Venus-Rab4 with NPS-2143. N = 12. (I–K) BRET between Nluc-mGq and Venus-Rab9 with cinacalcet, with (J) increasing cinacalcet concentrations, and (K) pEC50. N = 5. (L and M) BRET between Nluc-mGq and Venus-Rab9 in cells with increasing concentrations of cinacalcet, with (M) pEC50. N = 5. (N) BRET between Nluc-CaSR and Venus-Rab9 with NPS-2143. N = 12. (O) BRET between Nluc-mGq and Venus-Rab9 with NPS-2143. N = 11. (P–R) BRET between Nluc-mGq and (P) Rab5 ( N = 6), (Q) Rab4 ( N = 4), (R) Rab9 ( N = 5), in AP2σ-WT or AP2σ-R15H cells expressing CaSR with vehicle or cinacalcet. (S and T) Cinacalcet effect on responses in cells expressing a siRNA-resistant AP2σ-R15H plasmid combined with AP2σ-siRNA to knockdown endogenous protein or an AP2σ-R15H plasmid. N = 5. Statistics show: WT vs. AP2σ-R15H (red), AP2σ-R15H with vehicle or cinacalcet (blue) in (P)–(R). Statistical analyses were performed using two-way ANOVA with Dunnett’s test in (E), and Sidak’s test in (H,) (N), (O), (P), (Q), (R), (S), and (T), and one-way ANOVA with Dunnett’s test in (C), (G), (K), and (M). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. (A), (D), and (I) show examples of BRET data. Data shows mean ± SEM.

Article Snippet: Rab4 (RAB4A) Human siRNA Oligo Duplex (Locus ID 5867) , Origene , Catalog. no. SR303946.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Knockdown